A quantitative method for four theaflavins and two methylated theaflavin derivatives in black tea leaves was developed by solid-phase extraction and a high-performance liquid chromatographic method with photodiode array detection. The theaflavins in black tea leaves were extracted three times with 40 vol 50% aqueous ethanol (mg dry tea powder/mL) containing 2% ascorbic acid. The ethanol extracts were diluted 4-fold with distilled water. All diluted extracts were directly applied to the solid-phase C18 cartridge column without concentration. The fraction of theaflavins was obtained by 40% ethanol extraction after rinsing with water followed with 15% ethanol extraction. An aliquot of theaflavins after concentration was injected onto an ODS C18 reversed-phase column, and four theaflavins and two methylated theaflavins were sufficiently separated by a linear gradient system using distilled water and acetonitrile with 0.5% acetic acid. This analytical method is sensitive for the determination of a small amount of methylated theaflavins, since various interfering substances produced during the fermentation process were eliminated in advance by solid-phase extraction. Using this analytical method, we also demonstrated that methylated theaflavins were easily produced during the manufacture of black tea.
We investigated the active components responsible for the anti-allergic effect of Hypsizigus marmoreus on an oxazolone-induced type IV allergy in male ICR mice. Following both an oral administration at a dose of 250 mg/kg body weight and percutaneous administration at 0.10 mg/ear, significant anti-allergic activities were found in the 80% ethanolsoluble fraction (Fr. 1), and the low molecular weight fraction (Fr. 2, below a molecular weight of ca. 12000; Fr. 4, below ca. 3000) prepared from Fr. 1. These fractions included ergosterol, mannitol, and trehalose as antioxidants and/or anti-inflammatory components. The anti-allergic activities of these compounds and methionine were equal to or weaker than those of the fractions. Therefore, the molecular weights of the major active components in Hypsizigus marmoreus are thought to be less than ca. 3000, though ergosterol, mannitol, trehalose, and methionine would not be the main active components.
We investigated the antioxidant activities of two methylated theaflavins, theaflavin 3-O-(3-O-methyl) gallate (TF3MeG) and theaflavin 3-O-(3-O-methyl)gallate 3 -O-gallate (TF3MeG3 G), which were prepared from epigallocatechin-3-O-(3-O-methyl) gallate(EGCG3 Me) in the presence of burdock root-polyphenol oxidase (PPO). Formation of the methylated theaflavins was maximum at 30∼60 min of enzymatic reaction time, and decreased thereafter. The results suggest that the formation rate of methylated theaflavins in tea leaves can be increased by adjusting the PPO reaction during the fermentation process. The radical scavenging activities of TF3MeG and TF3MeG3 G were lower than those of the corresponding non-methylated theaflavins for peroxyl or DPPH radicals. However, the activity of TF3MeG3 G was much higher than that of the starting material (EGCG3 Me). The lipid solubility, estimated by 1-octanol /water partition coefficient, was in the order of TF3MeG3 G>TF33 diG≥TF3 G≥TF3MeG >TF3G≥TF. The lipid solubility of TF3MeG3 G was much higher than those of the other theaflavins and catechins.
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